Connexins (Cx) are a family of proteins essential for cell-to-cell communication and for the communication of cells with their environment. Cx43 is the most abundant connexin expressed in bone cells. The importance of Cx43 expression in the skeleton has been established by the demonstration of the osteoblast dysfunction and delay ossification in embryos of Cx43 null mice. However, the role of Cx43 and, in particular Cx43 hemichannels expressed in unopposed cell membranes, is far from being completely understood. In studies leading to this application it was demonstrated that bisphosphonates, besides stopping osteoclast-mediated resorption, might preserve bone strength at least in part by promoting osteoblast and osteocyte viability and that Cx43 expression is required for this survival effect. Thus, inhibition of apoptosis by bisphosphonates requires opening of Cx43 hemichannels, activation of the extracellular signal-regulated kinases (ERKs) and extra-nuclear retention of the kinases due to their interaction with the scaffolding protein (-arrestin. Cx43 is also required for prevention of osteoblast apoptosis by parathyroid hormone (PTH) in vitro; and the anabolic response to intermittent administration of the hormone is blunted in mice deficient mice in Cx43. In addition, Cx43 might be also involved in mechanotransduction in the skeleton. Thus, mechanical stimulation increases Cx43 expression, gap junction communication and hemichannel opening in osteoblasts and osteocytes. Based on this evidence, it is hypothesized that Cx43 is a crucial component of signaling pathways activated by pharmacologic, hormonal and mechanical stimuli in osteoblasts and osteocytes, via its involvement in hemichannels or gap junctions or its scaffolding properties. This hypothesis will be tested by a combination of in vitro and in vivo approaches. Studies in Aim 1 will examine the in vivo relevance of the Cx43/(-arrestin/ERK pathway for bisphosphonate actions on bone using bisphosphonate analogs that selectively activate this pathway; and they will delineate the mechanism by which association of Cx43, ERKs and (-arrestin leads to retention of ERKs outside the nucleus and the internalization of Cx43. Studies in Aim 2 will determine the role of Cx43 in the anti-apoptotic effect of PTH in vivo by examining whether the lack of anabolic effect of intermittent PTH administration to mice deficient in Cx43 is due to the inability of PTH to prevent osteoblast apoptosis. Aim 3 will define the role of Cx43 hemichannels in mechanotransduction in vivo and in vitro. This will be accomplished by investigating whether changes in gene expression and the anabolic response to mechanical loading are altered in Cx43 deficient mice; and whether Cx43 is required for the effects of mechanical stimulation in osteoblasts and osteocytes in vitro. We expect that this work will provide opportunities to improve the treatment of diseases with increased bone fragility. PUBLIC HEALTH RELEVANCE: These studies will advance our understanding of the role of Cx43 in the response of the skeleton to pharmacotherapeutic, hormonal and mechanical stimuli. We expect that this work will provide opportunities to improve the treatment of diseases with increased bone fragility.